Amyloid-β peptide (Aβ) is central to the pathology of Alzheimer's disease; it is the main constituent of brain parenchymal and vascular amyloid. Aβ extracted from senile plaques contains mainly Aβ1-140 and Aβ1-42, while vascular amyloid is mainly Aβ1-39 and Aβ1-40. The major soluble form of Aβ which is present in the blood, cerebrospinal fluid (CSF), and brain is Aβ1-40. Soluble Aβ which is circulating in the blood, CSF, and brain interstitial fluid (ISF) may exist as free peptide and/or associated with different transport binding proteins such as apolipoprotein E (apoE), apolipoprotein J (apoJ), transthyretin, other lipoproteins, albumin, and alpha2-macroglobulin (α2M).
LRP-1 binds amyloid β-peptide (Aβ) precursor protein (APP), apolipo-protein E (apoE) and α2-macroglobulin (α2M), (Herz and Strickland, 2001). But the exact biochemical mechanism(s) by which LRP-1 contributes to the onset of neurotoxic Aβ accumulations is unclear. LRP-1 binds secreted APP and influences its degradation (Kounnas et al., 1995) and processing (Pietrzik et al., 2002) leading to increased Aβ production (Ulery et al., 2000). It also mediates endocytosis of α2M-Aβ complexes in fibroblasts (Narita et al., 1997; Kang et al., 2000) and of apoE-Aβ and α2M-Aβ complexes in neurons in vitro (Jordan et al., 1998; Qiu et al., 1999). Overexpression of functional LRP-1 minireceptors in neurons of Alzheimer's PDAPP mice results in an age-dependent increase of soluble Aβ in the brain (Zerbinatti et al., 2004), which suggests that LRP-1 on neurons in vivo does not mediate Aβ clearance from brain.
Peripheral Aβ binding agents, e.g., an anti-Aβ antibody (DeMattos et al., 2002a), a soluble form of the receptor for advanced glycation endproducts, sRAGE (Deane et al., 2003) and/or ganglioside M1 and gelsolin (Matsuoka et al., 2003), rapidly clear Aβ from brain in vivo in various transgenic APP over-expressing mice. The idea that LRP-1 along the brain capillary membranes is a major clearance mechanism for Aβ in vivo has been. supported by findings demonstrating that intracerebrally infused Aβ1-40 undergoes rapid LRP-1-mediated transcytosis across the blood-brain barrier (BBB) (Shibata et al., 2000). Several questions, however, regarding a possible role of LRP-1 (including Aβ1-40, Aβ1-42, and mutant versions thereof) as a cargo/clearance receptor for brain Aβ remained unanswered. Whether Aβ is a direct ligand for LRP-1 initiating its own efflux from brain through interaction with the receptor at the BBB is not known. Reduced levels of LRP-1 in the brain were found in AD (Kang et al., 1997; Kang et al., 2000; Shibata et al., 2000). Whether high extracellular Aβ accumulations affect LRP-1 expression at the Aβ clearance site(s) in the brain is riot known.
But it was not previously demonstrated that low-density lipoprotein receptor related protein-1 (LRP-1) binds directly to Aβ. Cell surface receptors such as the receptor for advanced glycation end products (RAGE), scavenger type A receptor (SR-A), LRP-1, and low-density lipoprotein receptor related protein-2 (LRP-2) bind Aβ at low nanomolar concentrations as free peptide (e.g., RAGE, SR-A), and/or in complex with apoE, apoJ, or α2M (e.g., LRP-1, LRP-2). But it was not previously demonstrated that a soluble derivative of LRP-1 is able to directly bind Aβ in a bimolecular interaction.
WO 01/90758 and U.S. patent application Ser. No. 10/296,168 describe LRP-1's role in mediating vascular clearance of Aβ from the brain. It was taught that increasing LRP-1 expression or its activity can be used to remove Aβ and thereby treat an individual with Alzheimer's disease or at risk for developing the disease. A direct interaction between LRP-1 and Aβ was not described, nor was it taught or suggested that the two molecules are able to bind in solution without another ligand of LRP-1 such as apoE, apoJ, α2M, transthyretin, other lipoproteins, albumin, or RAP.
Here, it is demonstrated that LRP-1 and Aβ directly interact with each other (i.e., the two molecules are sufficient by themselves to specifically interact with each other) and this interaction on brain capillary membranes regulates retention of high B-sheet content neurotoxic Aβ1-42 and vasculotropic mutant Aβ while clearing Aβ1-40. LRP-1 mediates differential efflux of amyloid β-peptide isoforms from brain. Aβ1-40 binds to an immobilized LRP-1 fragment containing clusters II and IV with high affinity (Kd=0.6 nM to 1.2 nM) compared to Aβ1-42 and mutant Aβ. LRP-mediated Aβ clearance and transport across the blood-brain barrier in mice are substantially reduced by high B-sheet content in Aβ and deletion of the receptor-associated protein gene. Despite low Aβ production in the brain, transgenic mice expressing low LRP-1-clearance mutant Aβdevelop robust Aβ accumulations in the brain earlier than Tg-2576 Aβ-over-producing mice. At pathological concentrations (>1 μM), Aβ promotes LRP-1 degradation in brain endothelium consistent with reduced LRP-1 brain capillary levels observed in Aβ-accumulating transgenic mice, AD and patients with cerebrovascular β-amyloidosis. Thus, low affinity LRP-1/Aβ interaction and/or loss of LRP-1 at the BBB mediate brain accumulation of neurotoxic Aβ.
Receptor-associated proteins and receptor-mediated cell signaling are not required. Deletion of the RAP gene (Van Uden et al., 2002) which is associated with greatly reduced LRP-1 expression in the brain and at the BBB, but not deletion of the genes for the VLDL receptor or the LDL receptor, almost completely precluded rapid efflux of A from brain. Consistent with the findings here, LRP-1 levels were substantially reduced in brain microvessels in situ in a transgenic Aβ-accumulating animal model and patients with AD and cerebro-vascular β-amyloidosis.
New and nonobvious pharmaceutical and diagnostic compositions, and methods of treatment and diagnosis are taught herein to be applicable to the formation of amyloid and its role in disease. Other advantages of the invention are discussed below or would be apparent to a person skilled in the art from that discussion.